The research described in this proposal will investigate the effects of acute and chronic cannabinoid exposure on the phosphorylation of intracellular proteins. The protein chosen to be studied is MARCKS. Utilization of medicinal cannabinoids for treatment of pain is hindered by the risk of the development of tolerance and dependence. The mechanisms leading tolerance and dependence have not yet been fully elucidated. Using CB1 receptor-transfected human embryonic kidney cells (CB1-HEK) and membrane homogenates from the cortex, hippocampus, basal ganglia, striatum and cerebellum of Swiss-Webster mice, we will focus on the phosphatidylinositol cascade's role in the development of cannabinoid tolerance. Chronic stimulation of the CB1 receptor by delta-9 tetrahydrocannabinol (THC) or its analogs results in receptor desensitization, a point defined as tolerance. PKC inhibitors have been shown to reverse tolerance. Activation PKC with phorbol esters has been shown by others to translocate from the cytosol and phosphorylate MARCKS at the plasma membrane. Phosphorylated MARCKS can then translocate from the membrane to the cytosol. This research is designed to support the hypothesis that PKC phosphorylation of MARCKS occurs during the development of cannabinoid tolerance and that reversing tolerance by PKC inhibition attenuates phosphorylation and translocation of MARCKS.